2.11. Solid Phase Extraction (SPE) and HPLC Analysis of Plasma Polyphenols

The preparation of plasma samples for analysis of anthocyanins, phenolic acids and their metabolites was performed as previously described [38,44,45] with modifications. In brief, plasma (500 µL) samples were diluted by addition of 1 mL of 0.5% glacial acetic acid. To this, 50 µL of 100 µM 3-(4-Hydroxy-3-methoxyphenylpropionic acid), 3hmPA, was added as a recovery standard. The mixture was vortexed and centrifuged at 17,000× g, 4 °C for 15 min. The supernatant was loaded onto an Oasis HLB 60 mg 3 cc cartridge (Waters Limited, Elstree, Hertfordshire, UK) earlier conditioned by sequential addition of 1 mL each of acidified methanol (0.1% Trifluoroacetic acid, TFA, pH 2, in methanol) and acidified water (0.5% glacial acetic acid in distilled water). The SPE cartridge was then washed by sequential addition of 3 mL acidified water (0.5% glacial acetic acid in distilled water) and 1 mL of 0.5% acidified water in 20% methanol (water:methanol:acetic acid, 79.5:20:0.5 respectively). The cartridge was then dried by applying a vacuum for 5 to 10 s. This was followed by elution of phenolics, into speedvac tubes containing 200 µL of acidified methanol (0.5% acetic acid in methanol), by adding 1 mL of acidified methanol (0.1% Trifluoroacetic acid, pH 2, in methanol) onto the solid phase extraction cartridge twice, and passing through the SPE cartridge gravitationally. Elusion was completed with the application of a vacuum for 5 to 10 s to maximise recovery.

The eluent was dried using a speedvac system (Thermo Fisher Scientific Inc. Basingstoke, Hampshire, UK) and redissolved with 200 µL of HPLC mobile phase A (0.1% formic acid in HPLC water). The redissolved eluent was then filtered through a 0.20 µm filter (Sartorius Stedim Biotech, Gottingen, Germany) into HPLC vial and ran on an Agilent 1100 series (Agilent Technologies Ltd. Cheadle, Manchester, UK). The HPLC system is equipped with a diode array detector and Nova-Pak C₁₈ column (250 × 4.6 mm; 4 µm particle size; 30 °C; Waters Limited, Elstree, Hertfordshire, UK). The mobile phase A is 0.1 % formic acid in HPLC water while mobile phase B contained 0.1% HPLC water in acetonitrile and were pumped through the column at 0.3 mL per minute. Samples (50 µL) were injected and separated using the following gradient system (min/% mobile phase A/%mobile phase B): 0/95/5, 1/95/5, 20/70/30, 25/20/80, 30/20/80, 31/95/5 and 45/95/5. The eluent was monitored by photodiode array detection at 280 nm (phenolic acids and their metabolites) and 520 nm (anthocyanins and their metabolites) with spectra of products obtained between 220–600 nm. Retention time and peak spectra were used to identify phenolic acids, anthocyanins and their metabolites in the samples through comparison with that of authentic anthocyanidin, anthocyanin (Sigma Aldrich, Gillingham, Dorset, UK) and phenolic acid (Extrasynthese, Genay, France) standards. Standard curves (R² ranges from 0.95 t0 0.99) prepared were used to quantify the identified anthocyanidins and phenolic acids in each sample. Loses due to extraction procedures were corrected with the recovery standard, 3hmPA.